Electrical excitability plays important roles in neuronal differentiation, and functions of action potentials and neurotransmitter-activated channels are increasingly well understood. However, less is known about calcium-permeable channels that are widely expressed at very early stages of neuronal differentiation. Spinal neurons in amphibian embryos exhibit spontaneous transient elevations of intracellular calcium at early stages of development prior to synapse formation, both in vivo and in dissociated cell culture. Calcium influx through the ion channels that generate these transients initiates signal transduction cascades that determine subsequent steps of neuronal differentiation. In recent work we have discovered three classes of spontaneous transient elevations of intracellular calcium and shown that they regulate distinct aspects of differentiation in a frequency dependent manner, prior to synapse formation: 1) Calcium spikes are generated by developmentally transient calcium-dependent action potentials and regulate expression of the neurotransmitter GABA in vitro. 2) Growth cone calcium transients are generated locally in the growth cone and regulate the rate of axon extension in vivo and in vitro. 3) Filopodial calcium transients are produced at the tips of filopodia and regulate growth cone turning in culture. The proposed research has four specific aims that address the function of three classes of calcium transients. The first two aims investigate regulation of neurotransmitter expression by local calcium transients in the growth cone lamellipodium and its function in substrate adhesion in vitro. The fourth aim investigates the function of another class of growth cone calcium transients in vitro that depends on filopodial contact with a target cell and may regulate differentiation of presynaptic terminals. Understanding the functions of embryonic excitability analyzed in the research outlined in this proposal are expected to provide the basis for understanding and preventing developmental disorders of the nervous system.